ABSTRACT The overarching goal of our research is to understand the mechanisms of helicases and polymerases in processes such as DNA replication, transcription, and role of RIG-I helicase in innate immunity. Our research has made major contributions to understanding how these molecular motors move on nucleic acids to catalyze DNA and RNA strand separation and synthesis. These insights can provide the basis for understanding and treatment of diseases caused by dysregulation or malfunction of these enzymes. The unifying approach is quantitative characterization of the enzymatic reactions using rigorous biochemical and biophysical methods such as transient state kinetics, single molecule kinetics, computational kinetic modeling, and crystallography. Integration of structural and functional studies allows development of a complete mechanistic picture. The elegantly simple phage T7 enzymes allowed us to probe replication reactions with unprecedented temporal and spatial resolution, to develop new biophysical tools that correlate structure with function, and to propose new mechanisms that serve as a basis for studying more complex mitochondrial replication and transcription enzymes. Mitochondrial DNA deletions caused by defects in mitochondrial helicase and DNA polymerase affect energy production and result in a wide variety of neuromuscular diseases. Hence, in depth understanding of the enzymatic mechanisms of the mitochondrial DNA enzymes are critically needed. Our research on T7 and mitochondrial DNA replication will address key gaps in understanding the structure of the replisome, the proofreading mechanism of the DNA polymerase, and the DNA recombination activities of mitochondrial DNA helicase Twinkle. Our research on mitochondrial DNA transcription will provide mechanistic insights into the initiation mechanism, roles of the transcription factors, and address challenges in solving the structure of the initiation complex. Recently, we ventured into investigating the roles of RNA helicases in innate immunity by biochemically and structurally characterizing the RIG-I family of helicases. The RIG-I family of helicases are the cytoplasmic detectors of RNA viral infections, e.g. Dengue fever, West Nile, influenza, and hepatitis C. Our research will address key gaps in understanding the essential role of RIG-I helicases in initiating innate immunity by identifying crucial viral RNA recognition features, how viruses evade detection, and mechanisms that activate RIG-I. We will also address challenges in understanding the role of ATPase in RIG-I activation. This research will provide the mechanistic framework to quantitatively model the reactions of replication, transcription, and pathogen recognition that will guide in the development of therapies for human diseases including cancer, antiviral, and antimicrobial agents.